Morphology and ITS sequences provide insights into the phylogeny of Tongoloa (Apiaceae) from China

Background Tongoloa is a genus comprising approximately 20 species, primarily distributed in the mountainous regions of southwest China. The insufficiency of specimen materials and morphological similarities among species render it a taxonomically challenging genus within the Apiaceae family. To elucidate the phylogenetic relationships and taxonomy of Chinese Tongoloa, this study utilized a total of 115 nrITS sequences, including 47 recently obtained sequences, for phylogenetic reconstruction. Results Phylogenetic relationships reconstructed from ITS sequences indicate that the East Asia Clade and the Komarovia Clade are sister groups, and Tongoloa belongs to the East Asia Clade. Species of Tongoloa are subdivided into 3 distinct groups, all sharing similar fruit morphologies and are clearly differentiated from related taxa. Several Tongoloa-like members classified under other genera are interpreted to be closely related to Tongoloa. Morphological and molecular data indicate that Tongoloa, Sinolimprichtia subclade and Chinese Trachydium subclade are separate yet genetically contiguous taxa. It is confirmed that Tongoloa zhongdianensis belongs to the Hymenidium Clade, while Sinocarum is classified within the Acronema Clade. Two new taxa are found in the Hengduan Mountains. Conclusion Tongoloa is a genus within the East Asia Clade of Apiaceae, and the phylogeny reconstructed based on ITS sequences divides it into 3 main groups. By integrating fruit morphology and molecular phylogenetic analyses, we preliminary clarified the intricate taxonomic relationships among Tongoloa and related taxa. These results provide valuable opportunities for a deeper understanding of the phylogeny of Tongoloa. Supplementary Information The online version contains supplementary material available at 10.1186/s12862-024-02292-5.


Background
Tongoloa H.Wolff [1] is a genus including about 20 species distributed in the mountainous regions of East Asia [2], especially in southwest China [3,4].Most species are confined to alpine meadows, grasslands, shrubs, and forests ranging from 2000 to 4500 m [1,3,[5][6][7].Owing to their distribution in remote mountainous areas with limited accessibility, few of the previously collected specimens have contained mature fruits, which are considered to hold crucial taxonomic value in Apiaceae species [8][9][10][11][12].Insufficient specimen materials and similar morphologies make this genus taxonomically challenging [13].Pan and Watson stated that the current taxonomic treatment of Tongoloa should be considered tentative [3].Most Chinese species are still not fully recognized, and the general delimitation between Meeboldia, Sinoodielsia, Tongoloa and Vicatia has been problematic and controversial [14].
Over the past 20 years, scholars have endeavored to reconstruct the phylogenetic framework of Apiaceae using molecular methods, aiming to establish a more objective and robust classification system [15][16][17][18][19].Valiejo-Roman et al. reconstructed the phylogeny of Apioideae (Apiaceae) from the Himalayas by incorporating 13 newly obtained nrITS (nuclear ribosomal internal transcribed spacer) sequences, and the findings indicated that Tongoloa is related to Trachydium simplicifolium W. W. Smith and Hansenia Turcz.[20].After enriching samples and molecular makers of Apioideae from China, Zhou et al. proposed that Tongoloa exhibits a close relationship with specific species of Sinocarum H. Wolff ex R. H. Shan & F. T. Pu, Pimpinella L., Trachydium Lindl.and Sinolimprichtia H. Wolff, all residing within the East Asia Clade [21,22].Nevertheless, Xiao et al. argued that the genus Sinocarum does not belong to the East Asia Clade [23].Pimpinella purpurea (Franch.)H. Boissieu was molecularly closely related to members of Tongoloa [24].It was found that T. zhongdianensis S.L. Liou may related to Hymenidium Lindl.[25].Unfortunately, previous molecular analyses often lacked convincing morphological evidence.Examining the vouchers of previous molecular data is rather difficult, as they are usually scattered across herbariums in different regions.
China is the primary distribution area of Tongoloa, with all known species and a diverse range of morphological types [3,26].Recent field surveys in the Hengduan Mountains have led to a new understanding of species diversity of this genus [27,28].However, a comprehensive phylogenetic analysis of Tongoloa has yet to be conducted.Owing to its moderate length and rapid mutation rate, the ITS sequence serves as an efficient tool for analysis and validates the accuracy of species morphological taxonomy [17,21,[29][30][31][32].Additionally, the ITS sequence boasts a remarkable sequencing success rate and offers a cost-effective solution, significantly contributing to the accumulation of extensive species sequences in the current public database.In this study, we adopted extensive sampling and selected the ITS sequence as the molecular marker for phylogenetic reconstruction.Furthermore, we integrated observing and comparative analyses of fruit morphology to determine a more rational phylogenetic relationship for this genus.

Taxon sampling
To examine the morphological variations of Tongoloa species, a comprehensive study was conducted on a diverse range of specimens preserved in herbariums A, B, CDBI, E, G, GB, GH, HNWP, K, KUN, LD, MW, NAS, NY, P, PE, SZ, US, W, and WUK.Samples were preferentially collected from or near the type localities of the specimens to ensure accurate and reliable identification.The species identifcation was undertaken by Lingjian Gui and Xingjin He (Sichuan University, Chengdu, China).The nomenclature of taxa was primarily based on Flora of China [3] and the updated checklist of Chinese Apiaceae [26].Voucher specimens were stored in the herbarium of Sichuan University (SZ) in Chengdu, China (Table 1).The fruits of Tongoloa and related taxa were observed and photographed using a Nikon SMZ25 stereomicroscope (Japan).

DNA extraction and sequencing
Total DNA was extracted from silica-dried leaves using the plant genomic DNA kit (CWBIO, Beijing, China).The extracted total DNA was stored in a refrigerator at -20°C.The primers ITS4: 5'-TCC TCC GCT TAT TGA TAT GC-3' and ITS5: 5'-GGA AGT AAA AGT CGT AAC AAG G-3' , designed by White et al., were selected for PCR amplification of the target sequence [33].PCR was performed in a 30 µl amplification system, comprising 2 µL total DNA, 9 µl water, 1.5 µL forward primer, 1.5 µL reverse primer and 15 µl Taq PCR Master mix (CWBIO, Beijing, China).All PCR amplifications were performed on a Geneamp PCR System 9700 (USA).The ITS amplification protocol involved an initial denaturation step for 2 min at 95 °C, followed by 35 cycles of 60 s at 94 °C, 45 s at 52.5 °C, and 60 s at 72 °C, and a final extension step of 7 min at 72 °C.Bidirectional sequences were assembled using SeqMan in DNASTAR 5.01 [34], with subsequent necessary manual inspection.The completed ITS sequences and the boundaries of ITS1 and ITS2 were determined by comparison with the ITS sequences of Tongoloa available on NCBI.

Sequence alignment and phylogenetic analysis
A total of 115 ITS sequences of Apiaceae were used for constructing the phylogenetic tree.47 ITS sequences  S1).The ITS dataset includes 48 species from the East Asia Clade, 7 species from the Komarovia Clade, 14 species from the Acronema Clade, 4 species from the Hymenidium Clade (Sinodielsia Clade), and 5 species from Pleurospermeae [17].All sequences were aligned using MAFFT [35].The aligned sequences were manually inspected and the phylogenetic analysis was conducted using maximum likelihood (ML) and bayesian inference (BI) methods.The optimal DNA model was selected in Mega7 [36], and a ML tree with 500 bootstraps was subsequently constructed.Mrbayes v3.2 [37] was employed to infer a BI tree with 5 million generations using the GTR + G model.The first 25% of the trees were discarded, and the remaining trees were utilized to calculate posterior probability (PP) statistics.Bootstrap (BS) and PP values were annotated on the branches of a ML tree.Jalview2 [38] was used to visualize sequence base variations.

Fruit characteristics
The fruit morphology and anatomical features of Tongoloa were studied.We found that these fruits are primarily wide oval with a cordate base, exhibiting filiform ribs, 3 vittae in each furrow, and 4 vittae on the commissure.The stylopodium is depressed, while the ventral surface of the endosperm is flat-concave (Fig. 1).We subsequently examined the fruit morphologies of genera that have recently been considered closely related to Tongoloa in molecular phylogeny [21,22].The results revealed that the fruits of Pimpinella purpurea (Fig. S2) and Trachydium variabile (Fig. S3) exhibit a cordate base, 5 filiform ribs, 3 vittae in each furrow, and 4 vittae on the commissure.Additionally, they possess a depressed stylopodium, and a ventrally flat-concave endosperm, featuring closely align with those observed in common Tongoloa species.
The morphological results support the conclusion that Pimpinella purpurea and Trachydium variabile may be closely related to Tongoloa [22].However, in contrast, the fruits of Sinolimprichtia alpina exhibit a long obovate shape with a narrowed base and a fluffy peel, and possess 6 vittae on the commissures (Fig. S1 A).These features are significantly different from those of Tongoloa.Similarly, the fruits of Trachydium souliei are obovate with a narrowed base and wing-like ribs, and also display 6 vittae on the commissures (Fig. S1 D), distinguishing them from Tongoloa fruits.The mature fruits of T. zhongdianensis are distinctly dissimilar to those of the genus Tongoloa (Fig. S4).

Phylogenetic analysis
The ML consensus tree and BI tree have similar topological structures.The East Asia Clade and the Komarovia Clade are sister groups with strong support.Phylogenetic analysis using both ML and BI methods divides Tongoloa into three clustered groups (Fig. 2), although some branches require additional reinforcement for stronger phylogenetic support.Group I includes only one species, T. gracilis.

Sequence comparative analysis
A comparative analysis was performed on the ITS sequences of 13 Tongoloa species and 14 other closely related species from the East Asia Clade.The ITS sequence of Pimpinella purpurea exhibits identical specific base variations at positions 457-460 bp, 533 bp, 537 bp, and 610 bp with 4 closest Tongoloa or Tongoloalike species (Fig. 3A).The ITS sequence of Trachydium variabile shows a strong similarity to that of closely related Tongoloa species, yet it displays notable variations at loci 108 bp, 484 bp, 500 bp and 605 bp (Fig. 3B).

Phylogeny and taxonomy of Tongoloa
As of now, ITS sequences remain the principal criterion for defining the major clades and tribes of the Apiaceae family [17].Furthermore, the processing of precursor rRNA transcripts is a remarkably conserved biological mechanism.The secondary structure of the ITS in nuclear rRNA transcripts is widely documented across eukaryotes, and this feature has been employed to facilitate phylogenetic comparisons [39].
Based on the most extensive molecular sampling of Tongoloa and its closest relatives to date, it has been confirmed that Tongoloa belongs to the East Asia Clade of Apiaceae [17].The internal taxa of Tongoloa have been tentatively divided into three groups, including common Tongoloa species and some Tongoloa-like species, with the exception of T. zhongdianensis.Pimpinella purpurea and Physospermopsis cuneata are classified under Tongoloa group II, while Trachydium variabile falls under Tongoloa group III (Fig. 2).Morphological studies reveal that most of the species within the three groups, including those temporarily listed in other genera, share similar characteristics.These features include conical roots, slender stems, 3-ternate/pinnate leaves, depressed stylopodium, flat petals, fruits with a broadly ovoid shape and cordate bases, 5 filiform ribs, 3 vittae in each furrow and 4 vittae on the commissure.Additionally, the seed faces exhibit a flat-concave appearance.These shared characteristics support the classification of Tongoloa as an independent genus.
Previous studies have revealed that the phylogenetic relationship between Tongoloa, Sinolimprichtia alpina, Trachydium simplicifolium, and Trachydium souliei remains uncertain [21,22].In the phylogenetic tree of this study, although the Tongoloa groups, the Sinolimprichtia subclade, and the Chinese Trachydium subclade are almost independent of each other, they are located within the same major clade (Fig. 2), indicating a close genetic relationship.We have newly added fruit morphology data and found that the fruits of the Sinolimprichtia subclade and Chinese Trachydium subclade usually possess distinctive wing-like ribs and a higher number of vittae on the commissure (Fig. S1), which are noticeably distinct from the features of Tongoloa.In recent studies on chloroplast genomes and ribosomal DNA, Sinolimprichtia alpina was identified as a likely ancient hybrid taxon [40].The potential hybridization may introduce ambiguity to the boundaries between Tongoloa and its closely related taxa.Furthermore, the ubiquity radiation of plants in the Hengduan Mountains presents challenges to the the division of many generic boundaries [41,42].In light of molecular phylogenetic analyses and distinctive fruit morphological traits, acknowledging Tongoloa as a separate genus appears to be a justifiable classification strategy.This approach not only reflects the unique evolutionary lineage of Tongoloa but also respects its discernible morphological identity, thereby contributing to a more accurate and refined understanding of its taxonomic placement within the family.
Despite our diligent efforts in this study, we are unable to obtain molecular sequences for three dubious Tongoloa taxa.The species T. pauciradiata H. Wolff, identifiable only through its type specimen collected in Lhasa, Tibet (barcode K000075377), exhibits fruit and plant morphologies similar to advanced Apioideae taxa, diverging from the primitive characteristics of basal lineages such as the East Asia Clade.Tongoloa rockii H. Wolff is represented solely by type specimens (barcodes E000000521 and GH00077982) from Yulong Snow Mountain in Yunnan.This species, characterized by its short and slender plants, thick root and 4-pinnate/pinnatifid leaves, is acknowledged as a good species by Fading Pu [43].After analyzing the morphological features and ITS sequences of similar species, it is evident that its phylogenetic position belongs to the Tongoloa group III.The species T. smithii H. Wolff (barcode GB-0048829) is exclusively found in its type locality, Maerkang, Sichuan.The distribution of this species lies within 200 km of T. elata's type locality in Huangshengguan, Songpan, Sichuan.Despite lacking fruits on its type specimen, T. smithii exhibits morphological similarities in its flowers, leaves, and overall plant structure to T. elata, indicating a possible close relationship or even species identity.
Zhou et al. provisionally referred to the Pimpinella species in the East Asia Clade as the Chinese Pimpinella subclade [21,22].However, the findings of this study have underscored the imperative need to further categorize these Pimpinella taxa into two separate subclades, temporarily designated as Chinese Pimpinella subclade A and Chinese Pimpinella subclade B (Fig. 2).Subclade A is phylogenetically distant from Tongoloa and exhibits a close relationship with Keraymonia.Currently known species include Pimpinella henryi Diels and P. rhomboidea Diels.The fruit bases of this subclade are cordate, and have 5 filiform ribs, exhibiting some similarity to Tongoloa.However, the stylopodium shape, style length, and the number of vittae in the fruit significantly differ from Tongoloa.Chinese Pimpinella subclade B stands as a pivotal groups in this study.Currently, the confirmed species primarily include P. purpurea, whose ITS sequences exhibit a close clustering with species of Tongoloa group II.Moreover, morphologically, Pimpinella purpurea exhibits an ovoid fruit shape with a cordate base, filiform ribs, 4 commissural vittae, and a flattened stylopodium (Fig. S2), all of which align closely with the morphological features of Tongoloa.
Trachydium variabile is presently a species lacking extensive morphological and molecular studies, and is not included in Flora of China [43].We verified the original literature, the type specimens, as well as fresh samples collected from the type locality.The newly collected samples exhibited a remarkable consistency with the type specimens.Additionally, we enhanced the population sample by incorporating specimens from the vicinity of Longriba, Hongyuan, Sichuan.The sequencing results reveal a congruency in the ITS sequences of both populations.The phylogenetic tree constructed on the basis of ITS sequences positioned T. variabile within Tongoloa group III.In terms of morphology, the flower, fruit, and leaf characteristics of this species are similar to those of the typical species of Tongoloa group III (Fig. S3).Analysis revealed that the ITS of Trachydium variabile had undergone significant genetic variation compared with related taxa, particularly with four unique base variations at the 108 bp, 484 bp, 500 bp, and 605 bp loci (Fig. 3B).These results hint that Trachydium variabile is an independent species in Tongoloa Group III.
Also noteworthy, the results of molecular analysis support the suggestion made by Pan and Watson in Flora of China, to classify Physospermopsis cuneata as belonging to Tongoloa.However, it is unfortunate that we failed to collect samples with mature fruits from the type locality of Physospermopsis cuneata in Yulong Snow Mountain, Yunnan.Therefore, it is crucial to supplement the collection materials for future research purposes.
The mature fruits of Tongoloa zhongdianensis collected from the type locality exhibit significant morphological differences from Tongoloa species (Fig. S4).ITS molecular markers indicate that the systematic position of this species falls within the Hymenidium Clade (Sinodielsia Clade), possibly having a closer relationship with some species of Meeboldia, while being distant from the East Asia Clade where Tongoloa resides.Currently, this taxon is still considered an independent species, but its precise taxonomic treatment requires further determination.
Sinocarum was previously considered to be closely related to Tongoloa [14].Valiejo-Roman et al. conducted a sampling and phylogenetic study of Apiaceae from the Himalayan region, involving one representative taxon of Sinocarum and another representative taxon of Tongoloa [20].The result indicates that despite the morphological similarities between Sinocarum and Tongoloa, they do not share a close genetic relationship.Recent research, bolstered by our findings, confirms that the majority of Sinocarum species, particularly S. coloratum, S. cruciatum (Franch.)H. Wolff, and S. vaginatum H. Wolff, all belonging to the Acronema Clade, exhibit significant genetic divergence from Tongoloa [23,44].The morphological similarity between these two genera may be attributed to convergent evolution resulting from selective pressures in high-altitude environments rather than genetic relationships.Indeed, there are significant morphological differences between the mature fruits of Sinocarum [23] and Tongoloa.Unfortunately, most of the previously gathered specimens of Sinocarum, including type specimens, were devoid of mature fruits, potentially leading to erroneous identifications in the past.

Two new species of Tongoloa
The morphological variation of Tongoloa species distributed in the Hengduan Mountains is complex, and specimens are easily misidentified when morphological characteristics are not sufficient [23,27,45].We found a questionable specimen [T.N. Ho (HNWP), B. Bartholomew (CAS), M. Watson (E) and M. Gilbert (MO at BM) 2685] preserved in the Qinghai-Tibetan Plateau Museum of Biology (Xining, Qinghai, China).It was identified as Tongoloa gracilis.Nevertheless, the petal tips of this particular plant are blunt instead of pointed, which is not aligned with the typical features of T. gracilis with incurved petal tips.In 2019, guided by sampling records, we conducted a field investigation to Jiangxi Forest Farm in Jiangxigou, Yushu, Qinghai Province, and collected the fresh materials of this unusual species on the grassy slope under Picea trees.The ITS phylogenetic analysis reveals that this species occupies a distinct branch within Tongoloa group II (Fig. 2).We conducted a thorough examination of all available specimens and literature, with a particular emphasis on the taxa from the Qinghai-Tibet Plateau.After a thorough analysis encompassing morphology, nrITS sequence, and geographic distribution, we have determined that this plant belongs to an undescribed species.Given the distinctive morphological characteristics of its petals, we have designated it as T. spathulata.
Xinlong County, situated in the Hengduan Mountains of Sichuan Province, boasts an altitude ranging from about 2,700 m to 5,900 m.The continuous mountains here are separated by several river valleys.There have been scant records of Apiaceae specimens in Xinlong County and its neighboring regions (at least until recently).In 2020, during our survey of plant resource in this County, we discovered an special species of Tongoloa.The plants preferentially thrive in humid places at altitudes of 3200-3800 m, growing among shrubs or in herb communities.Combined with the geographical distribution, we examined the morphology and molecular marker (ITS) of the newly collected samples (Figs. 2 and 3), and confirmed that this species is closely related to T. spathulata, which is distributed in Qinghai.However, the petals, fruits and ITS sequences of this species are significantly different from those of similar taxa, and it cannot be placed in any known Tongoloa or other Apiaceae species.Here we formally describe it as a new species, T. xinlongensis.
Etymology.The species is named after the spatulate petals of this plant.
Distribution and habitat.Tongoloa spathulata is currently found in Yushu, located on the eastern Qinghai-Tibet Plateau.It grows on slope, at an altitude of 3600-3900 m.Fl. and Fr.Aug.-Oct.

Conclusion
Tongoloa is a genus within the East Asia Clade of Apiaceae, and the phylogeny reconstructed based on ITS sequences divides it into 3 main groups.These groups have similar fruit morphology.By integrating fruit morphology and molecular phylogenetic analyses, we preliminary clarified the intricate taxonomic relationships among Tongoloa, Sinocarum, Chinese Pimpinella subclade, Sinolimprichtia subclade, and Chinese Trachydium subclade.Furthermore, we proposed 2 new species, thereby suggesting a potentially richer species diversity of Apiaceae in the Hengduan Mountains.

Fig. 3
Fig. 3 Comparison of aligned ITS sequences of 24 taxa from East Asia Clade.(A) Pimpinella purpurea shares common variations at loci 457-460 bp, 533 bp, 537 bp and 610 bp with other species in Tongoloa group II (indicated by purple font and black boxes); (B) The specific variations of Trachydium variabile, relative to closely related species, are found at loci 108 bp, 484 bp, 500 bp and 605 bp (indicated by blue font and red boxes)
and Hymenidium virgatum.Notably, while the Tongoloa groups, the Sinolimprichtia subclade and the Chinese Trachydium subclade are each recognized as separate taxa on the phylogenetic tree, their branching patterns reveal remarkably close affinities, suggesting complex speciation or recent divergence within the broader evolutionary context.Tongoloa zhongdianensis is not located within the East Asia Clade, but it exhibits a significant association with taxa belonging to the Hymenidium Clade.